The periplasmic phosphate binding protein is a product of the phoSgene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coll. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli.
These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding capacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The K D values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the KD value was found to be 9-31 times higher than the values obtained for the wild type and the phoT mutant. The apparent K m values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. Vm, x of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.
Both phoT and pstB mutants, which belong to different genes, meet these criteria. A comparative study of Pi binding to their periptasmic proteins has revealed that mutants in the pstB locus are impaired in the Pi-binding activity of their Pi-binding protein.
Materials and methods
Bacterial strains.
Table 1 lists the strains used, their genttype and source. All strains labelled EY were constructed in our laboratory.
Media, transduction, three point cross mapping, sexduction, complementation tests, assay of AP activity, preparation of periplasmic extracts were all done as described before (Levitz et al. 1984) fl-glycerol-P selective plates are described by Torriani and Rothman (1961).
Gel electrophoresis. Periplasmic extracts (30 gg proteins)
were run on non-denaturing polyacrylamide slab gels as described earlier (Yagil et al. 1975)